A number of new technologies have been developed from our work over the last few years that are now at the core of our discovery efforts in the areas of HIV/AIDS and cancer biology. Two advances are especially important for our work at the intersection of structural biology and virology. We extended our efforts in subvolume averaging of cryo-electron tomographic volumes by applying automated, iterative, missing wedge-corrected 3D image alignment and classification methods to distinguish multiple conformations that are present simultaneously. One useful recent biological application of our methods has been in the area of vaccine design in influenza to determine the extent to which HA stem regions on the surface of the virus are accessible to broadly neutralizing antibodies. To address this, we first obtained 3D structures from cryo-electron tomography of HA on intact 2009 H1N1 pandemic virions in the presence and absence of the antibody C179, which neutralizes viruses expressing a broad range of HA subtypes including H1, H2, H5, H6 and H9. By fitting previously derived crystallographic structures of trimeric HA into the density maps, we deduced the relative locations of the molecular surfaces of HA involved in interaction with C179. To separate any unliganded from antibody-bound HA spikes, we used a newly developed procedure for collaborative alignment. Our approach uses an iterative Joint Alignment-Classification (JAC) technique, in which the classical similarity measure based on pair-wise distances between two particles, or a particle and a class average, is replaced by a one-to-many collaborative similarity function measured between a particle and a group of particles. This collaborative alignment framework enables robust alignment of linearly correlated heterogeneous images. Our analysis shows that despite their close packing on the surface of the virus, 75% of all HA trimers are complexed with the antibody, establishing that universal influenza vaccines that elicit such antibodies could be effective in controlling the infection. In a different development, we have made significant inroads to develop the potential of obtaining near-atomic resolution structures using cryo-EM. Atomic resolution models for proteins and protein complexes are usually obtained using X-ray crystallography or NMR spectroscopy, and in selected instances, by cryo-EM of ordered protein assemblies. The vast majority of high resolution structures obtained using cryo-EM have been typically restricted to large, well-ordered entities such as helical or icosahedral assemblies or two-dimensional crystals. We have now shown that emerging methods in single-particle cryo-EM can be used for structure determination at near-atomic resolution, even for much smaller protein complexes with low symmetry, by determining the structure of the 465-kDa enzyme beta-galactosidase. Our 3.2 Angstrom structure for beta-galactosidase represents the highest resolution achieved so far by single particle cryo-EM for a protein complex smaller than 500 kDa. In recent developments, structures of a ribosome complex (more than 2000 kDa), metalloenzyme complex (1200 kDa), proteasome (700 kDa) and an ion channel (300 kDa) have been reported at resolutions of 3.2, 3.4, 3.3 and 3.4 Angstroms, respectively. The advances that we and others have now made in determination of structures of protein complexes and membrane proteins at near-atomic resolution mark a critical shift in structural biology. One reason for the excitement is the demonstrated prospect that at least in some instances, it may be possible to dispense with the complicated process of crystallizing proteins in order to determine their structure. Another reason is the fact that atomic resolution structures can be determined from just a few microliters of a protein suspension, and from as few as 12,000 molecular images. There are other technical firsts in our work. We present evidence for the first time for the selective susceptibility of negatively charged residues to radiation damage that occurs with exposure to electron beams. We show that fractionating the electron dose allows one to maximize the level of structural detail that is visualized in density maps obtained by cryo-electron microscopy. Thus, using only the first few exposures enabled determination of maps where the densities of all side chains could be visualized clearly, before the selective degradation of density from negatively charged residues. Further, our studies show that atomic resolution structures can be obtained even from regions in proteins that are normally altered or deleted to obtain diffracting crystals, and provide a new dimension for the use of hybrid approaches for structure-function studies of intact, full-length protein complexes. Two other advances that helped achieve high resolution were improvements in methods for compensation of drift and beam induced motion during the electron exposure, and in more accurate determination of contrast transfer function (CTF) parameters, as discussed below. For drift correction, the cumulative averages of previously aligned frames were used as a reference to align individual frames of each movie by cross-correlation. The increased signal-to-noise ratio of the cumulative average of frames which was used as a reference to align the raw frames, results in significant improvements in the accuracy of motion correction as evidenced by better defined Thon rings in the drift-corrected images. Accuracy of CTF determination is one of the main factors that impact resolution in single-particle cryo-EM especially in the near-atomic resolution regime. Taking advantage of the capability of direct electron detectors to produce dose fractionated movies, we show that in addition to providing opportunities for motion correction and mitigation of radiation damage effects, movies also provide a way to improve the accuracy of CTF determination and therefore the resolution of single-particle reconstructions. The way we accomplish this is by estimating the parameters of the CTF of each micrograph using radially averaged power spectra obtained by periodogram averaging with tiles extracted from all frames of each movie. Compared to the standard approach of using tiles extracted from the average of frames (after drift-correction), our method does not require frames to be pre-aligned and results in better-defined 1D CTF profiles allowing more accurate determination of defocus. The improvement in the resolvability of zero-crossings obtained using this approach allowed us to set the upper resolution limit for CTF estimation at 3 Angstromwhile still providing reliable CTF fits. The prospect that the determination of protein structures to atomic resolution will no longer be limited by size or by the need for crystallization represents a significant and exciting horizon in structural biology. Rather than simply using cryo-EM maps, typically in the 6 - 20 Angstrom resolution range, as an envelope in which to fit structures obtained by X-ray crystallography, there is the exciting prospect of using cryo-EM to derive de novo, high-resolution structural models of proteins in one or multiple functional conformational states. The stage is now set for the application of these methods to analyze structures of a wide variety of biologically and medically relevant multi-protein complexes and membrane protein assemblies, which have historically represented the most challenging frontier in structural biology.